Herpetology Notes, volume 12: 873-876 (2019) (published online on 14 August 2019)

Notes on a little known Central African Reed , schoutedeni Laurent, 1943

Abraham Bamba-Kaya1, Ange-Ghislain Zassi-Boulou2, Elie Tobi3, T. Danielle Hayes4, Daniel M. Portik5, David C. Blackburn4, and Gregory F. M. Jongsma4,*

The Hyperolius is composed of colourful in WGS84). Laurent (1946) later reported additional treefrogs that are widely distributed across sub-Saharan male and female specimens from Buta and Monga in Africa. This large genus (>150 species) contains small northern DRC. In 1975, Arne Schiøtz collected the first to medium-sized that are often similar in external records from Republic of the Congo in Nsele, Pool morphology yet highly variable in colouration and Department (-4.083°S, 15.050°E; Schiøtz, 2006). Inger pattern (Schiøtz, 1999; Portik et al., 2019). In addition, (1968) previously reported H. schoutedeni from similar species often display varying degrees of sexual size sites of marshes and ponds in Garamba National Park dimorphism and colour variation between the sexes in northeastern DRC. While H. schoutedeni is believed (sexual dichromatism; Bell and Zamudio, 2012). to be widespread across the Congo Basin (Laurent, Together, these patterns of variation make species 1950; Schiøtz, 2006; Figure 1), its resemblance to the identification notoriously difficult among many species congener H. cinnamomeoventris has led to uncertainty of hyperoliid frogs. For this reason, we have taken an in understanding its precise distribution. Based on its integrative approach to confirm any new geographic known distribution, H. schoutedeni likely also occurs records for this genus by combining morphology, in Central African Republic and southern Sudan (Frost, advertisement calls, and genetics (e.g., Portik et al., 2019). Despite its range and close proximity to Uganda, 2016; Jongsma et al., 2017). Here we report the first Schiøtz (1975, 2006) reported no evidence of any country record of Hyperolius schoutedeni from Gabon specimens from this country. and the second record for Republic of the Congo. In 2018, ABK, AGZB, ET, and GFMJ surveyed Hyperolius schoutedeni was described by Laurent at two sites on the Batéké Plateau in (1943) based on a single female specimen collected southeastern Gabon and central Republic of the Congo. by Dr. Henri Schouteden (1881–1972) in Kunungu in western Democratic Republic of Congo (DRC; georeferenced to -2.100°S, 16.433°E; all localities given

1 Institut de Recherches Agronomiques et Forestières (IRAF), Centre National de la Recherche Scientifique et Technologique (CENAREST), BP 842, Libreville, Gabon. 2 Institut national de Recherche en Sciences Exactes et Naturelles (IRSEN), Brazzaville, Republic of the Congo. 3 Gabon Biodiversity Program, Smithsonian Conservation Biology Institute, Gamba, Gabon. Figure 1. Map of known H. schoutedeni records. Squares 4 Florida Museum of Natural History, University of Florida, denote published records, circles denote unpublished records. Gainesville, FL, USA. The type locality, Kununga, DRC, is represented by a star. The 5 Department of Ecology and Evolutionary Biology, University IUCN distribution of H. schoutedeni is shown in translucent of Arizona, Tucson, Arizona, USA. blue and H. cinnamomeoventris in translucent green (IUCN, * Corresponding author. E-mail: [email protected] 2019). 874 Abraham Bamba-Kaya et al.

On the evenings of 16 and 18 May 2018, they collected cinnomeomeoventris from Solonga National Park, DRC 10 H. schoutedeni from a flooded plain in Ejangoulou (Schick et al., 2010). According to Schiøtz (1999), H. Village, Plateaux Department, Haut-Ogooué Province, schoutedeni is distinguishable from the similar and Gabon (-1.4943°S, 14.4162°E). On 24 and 28 May 2018, syntopic H. cinnamomeoventris by lacking sexual they collected 7 individuals in large flooded savannahs dichromatism, meaning that females and males of near Lékéty Village, Cuvette Department, Republic of H. schoutedeni are similar in colour and pattern and the Congo (1.5734°S, 14.9477°E; 1.5858°S, 14.9290°E). resemble the male colouration of H. cinnamomeoventris At both sites, H. schoutedeni and H. cinnamomeoventris (Figure 2). In addition, H. schoutedeni is distinguishable were found at similar perch sites on grasses and reeds 10 by having a longitudinal stripe on the dorsal side – 100 cm above the water. Our collections represent the of the crus (i.e., the portion of the leg containing the first record of Hyperolius schoutedeni in Gabon and the tibiofibula), which differs from the uniform or darkly second record for Republic of the Congo. These extend spotted crus of H. cinnamomeoventris (Inger, 1968; the species distribution by ~250 km west of the nearest Schiøtz, 1999; Figure 2A, B). known locality in DRC. Inger (1968) reported that H. schoutedeni in life from Hyperolius schoutedeni is a small, beige reed frog with Garamba often have red spots on the dorsal surface of the yellow dorsolateral lines, and both sexes share this colour foot; however, this was not observed in any individuals pattern (Figure 2A). Female H. cinnamomeoventris are collected in 2018 (Gabon or Republic of the Congo) bright green in colouration and readily distinguishable nor was it noted by Schiøtz (2006) or Laurent (1943, from males (Figure 2B). Interestingly, the two female 1946). This may be a unique feature of the populations H. cinnamomeoventris encountered in Lékéty, Republic at the northeastern extent of the range of this species of the Congo were not green and instead appeared as these spots are not recorded in any other collection more like a larger version of the male form (Figure locality. In H. schoutedeni, adults and juveniles retain a 2B). Genetically, these individuals are closest to H. yellow mid-dorsal stripe that is outlined in black. Male

Figure 2. Hyperolius cinnamomeoventris (top panel) and H. schoutedeni (bottom panel), highlighting distinguishable characters of these two species. Column A are males. Column B are females including typical females including a typical female H. cinnamomeoventris (green) from the type locality, Kalandula Falls, Angola (UF:Herp:185651) and an aberrant, non-dichromatic female from Republic of Congo. Column C contains photos of vocal sacs of the two species. Notes on a little known Central African Reed Frog 875

H. schoutedeni have a large and granular gular gland procedures for Qiagen DNeasy kits. Sequence data were that covers the entire space between the lower jaw obtained from the mitochondrial marker 16S ribosomal (Inger, 1968) in contrast to the smaller gular gland of RNA (16S) using primers 16SA-l and 16SB-h (Palumbi male H. cinnamomeoventris (Figure 2C). Hyperolius et al. 1991). We performed polymerase chain reactions balfouri, another savannah-specialist that was recently (PCRs) using 20ul volume consisting of: 2ul DNA discovered in Gabon (Jongsma et al., 2017) and also has and AmpliTaq Gold master mix. The amplification a large granular gular gland. However, this species lacks process for 16S was as follows: denaturation at 95°C the small black points that speckle the throat and gland for 10 minutes, then 35 cycles of 96°C for three of H. schoutedeni (Figure 2C). seconds, 54°C for three seconds, 68°C for 15 seconds, The advertisement call of H. schoutedeni is described and a final extension at 72°C for 10 seconds. We as a double-click (Schiøtz, 2006), which matches our checked the success of the PCR by performing a gel recording of a male frog in Gabon (UF:Herp:185889; electrophoresis before plating and sending the samples ML 236287; Figure 3A). Each pair of clicks in the call to Genewiz in Pennsylvania. To date, there have been of H. schoutedeni is spaced approximately 1.75 seconds no published DNA sequence data for H. schoutedeni. apart and is easily distinguishable from the quick series Using a database of >1,300 16S sequences of clicks emitted by H. cinnamomeoventris that are (Portik et al. 2019), we performed a BLAST search spaced about 0.2 seconds apart (Schiøtz, 1999; Figure and established that no individuals identified in the 3B). This distinct double-click is distinguishable from field as H. schoutedeni have high sequence similarity other savannah-specialists that could have overlapping to individuals of H. cinnamomeoventris. Instead, these distributions with Hyperolius schoutedeni, including are part of a clade H. schoutedeni with H. kivuensis, H. H. adspersus, H. balfouri, H. kivuensis, H. lateralis, balfouri, and H. quinquevittatus. A close relationship and H. viridiflavus. Recordings were made by GFMJ with H. kivuensis was previously suggested by Laurent on a Pixel phone (44 kHz sample rate) in the field and (1943, 1946), based on similarities of foot-webbing analysed using the R package seewave (R Core Team and the position of the nares. While clearly divergent 2018; Sueur et al., 2008). from H. cinnamomeoventris, robust geographic and Finally, we sequenced a fragment of the 16S taxonomic sampling of Hyperolius across Central mitochondrial gene of all specimens of Hyperolius Africa is needed to evaluate the phylogenetic affinities collecting during our work in Gabon and Republic of of H. schoutedeni. Congo. Genomic DNA was extracted following standard

Figure 3: A) Advertisement call of H. schoutedeni (UF:Herp:185886) and (B) H. cinnamomeoventris (UF:Herp:185115). 876 Abraham Bamba-Kaya et al.

The specimens used in this work are accessioned at the Laurent, R.F. (1943): Les Hyperolius (Batraciens) du Musée Florida Museum of Natural History (FLMNH, University Congo. Annales du Musée Royal du Congo Belge, Sciences of Florida), the Gabon Biodiversity Collection in Zoologiques 4: 61–140. Laurent, R. F. (1946): Note sur une petite collection de Megalixalus Gamba, and Institut national de Recherche en Sciences et d’Hyperolius de l’Uélé et sur Hylambates gresshoffii. Bulletin Exactes et Naturelles in Brazzaville. All 16S sequences du Musée Royal d’Histoire Naturelle de Belgique 22: 1–16. of H. schoutedeni and H. cinnamomeoventris from our Laurent, R. (1950): Genres Afrixaḻus et Hyperolius Amphibia sampled sites are available on GenBank (MK544949 salientia. Exploration of National Park Albert, Mission G. F. – MK544969; Supplementary material table S1) and the de Witte (1933–1935). Institut des Parcs Nationaux du Congo advertisement call is available on the Macaulay Library Belge 64: 1–120. (ML 236287). The same specimen that was recorded Palumbi, S. R., Martin, A., Romano, S., McMillan, W. O., Stice, L., Grabowski, G. (1991): The Simple Fool’s Guide to PCR. (UF:Herp:185889) was CT scanned and accessioned on Department of Zoology, Special Publication, University of MorphoSource (doi: 10.17602/M2/M83156) Hawaii, Honolulu, 44 pp. Portik, D.M., Jongsma, G.F., Kouete, M.T., Scheinberg, L.A., Acknowledgements. We thank Institut National de Recherche Freiermuth, B., Tapondjou, W.P., Blackburn, D.C. (2016): A en Sciences Exactes et Naturelles (IRSEN) for issuing collection survey of amphibians and reptiles in the foothills of Mount Kupe, authorizations in the Republic of Congo (No. 170 / MRSIT Cameroon. Reptile Conservation 10(2): 37–67. / IRSEN / DG / DS) and the Centre National de la Recherche Portik, D.M, Bell, R.C., Blackburn, D.C., Bauer, A.M., Barratt, Scientifique and Technologique (CENAREST) for authorizations C.D., Branch, W.R., Burger, M., Channing, A., Colston, in Gabon (AR0016 / 17 / MESRS / CENAREST / CD / CST / T.J., Conradie, W., Dehling, J.M., Drewes, R.C., Ernst, R., CSAR). Field work was supported by the Florida Museum of Greenbaum, E., Gvoždík, V., Harvey, J., Hillers, A., Hirschfeld, Natural History (to DCB) and the Smithsonian Biodiversity M., Jongsma, G.F.M., Kielgast, J., Kouete, M.T., Lawson, L.P., Program. We are thankful to the village chiefs in Edjangoulou, Leaché, A.D., Loader, S.P., Lötters, S., Van Der Meijden, A., Gabon (Thomas) and Lékéty, Republic of Congo (V. Ekobanga) for Menegon, M., Müller, S., Nagy, Z.T., Ofori-Boateng, C., Ohler, helping facilitate our field work. We thank R. Hedley for analyzing A., Papenfuss, T.J., Rößler, D., Sinsch, U., Rödel, M-O., Veith, call data, D. Meirte at the Royal Museum for Central Africa for M., Vindum, J., Zassi-Boulou, A-G., McGuire, J.A. (2019): providing photos of type material, M. Medler and M. Young Sexual Dichromatism Drives Diversification within a Major for cataloging our recording in the Macaulay Library (Cornell Radiation of African Amphibians. Systematic Biology. https:// University), E Stanley at the University of Florida for CT-scaning doi.org/10.1093/sysbio/syz023. material, and A. Feistner for thoughtful comments on the first R Core Team (2018): R: A language and environment for statistical draft of this note. This publication is the Smithsonian Institution, computing. R Foundation for Statistical Computing, Vienna, Center for Conservation and Sustainability (CCS), Gabon Austria. URL https://www.R-project.org/. Biodiversity Program contribution number 183. 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